Subcellular Localization of a Protein Kinase Required for Cell Cycle Initiation In Saccharomyces Cerevisiae: Evidence for An Association Between the CDC28 Gene … (original) (raw)
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Journal of Cell Biology, 1987
The product of the Saccharomyces cerevisiae gene CDC28, a protein kinase required for initiation of the cell division cycle, was localized within yeast cells. By using immunofluorescence methods, the CDC28 product was shown to be primarily cytoplasmic in distribution. The gene product was localized largely to the particulate fraction by differential centrifugation after mechanical disruption in aqueous buffers. The particulate association was not affected by the presence of nonionic detergent. To refine this localization further, a procedure was developed for the preparation of yeast cytoplasmic matrices which resemble the cytoskeletons of vertebrate cells on the basis of methodology, immunochemistry, and gross ultrastructure. A portion of the CDC28 product was found to be tightly associated with these detergent-insoluble cytoplasmic matrices by both immunoflourescence and immunoblotting procedures. Although, for technical reasons, precise quantitation was not possible, it is estimated that a minimum of 2-15 % of the total CDC28 product pool is involved in the association with the insoluble matrix. Alcohol dehydrogenase, a soluble cytoplasmic protein, was found not to be associated with the cytoplasmic matrices at any detectable level, whereas, in contrast, ,o10--40% of the total cellular actin, a bonafide cytoskeletal protein, was present in these structures. The proportion of CDC28 gene product associated with the particulate fraction, and perhaps the insoluble matrix, appears to be substantially decreased during the preparation of spheroplasts.
Mitotic Role for the Cdc28 Protein Kinase of Saccharomyces cerevisiae
Proceedings of The National Academy of Sciences, 1990
The Cdc28 protein kinase functions in the G, to S phase transition of the cell cycle of the budding yeast Saccharomyces cerevisiae. This is in contrast with observations of the homologous protein kinase from a variety of metazoans, where activity and function are associated with the G2 to M
Methods in molecular biology (Clifton, N.J.), 2011
Methods are described here to monitor changes in protein level and subcellular localization during the cell cycle progression in the budding yeast Saccharomyces cerevisiae. Cell synchronization is achieved by an α-factor-mediated block-and-release protocol. Cells are collected at different time points for the first two cell cycles upon release. Cellular DNA contents are analyzed by flow cytometry. Trichloroacetic acid protein precipitates are prepared for monitoring levels of cell cycle regulated proteins by Western blotting. The dynamic changes in protein subcellular localization patterns are examined by indirect immunofluorescence microscopy.
Fission yeast cyclin: subcellular localisation and cell cycle regulation
Journal of Cell Science, 1989
Entry into mitosis in the fission yeast Schizosaccharomyces pombe involves the interaction of a number of genes with the major cell cycle control gene, cdc2+. One of these, cd cl3 +, encodes a protein with homology to cyclin. By indirect immunofluorescence microscopy using antibodies to the appropriate bacterially-expressed protein, we have shown that both cdcl3 and cdc2 are nuclear proteins in S. pombe. Both are localised to a nuclear domain distinct from that occupied by the DAPI-staining chromatin. The immunofluorescence signals of both proteins show a progressive increase during interphase but are undetectable at mitosis. Loss of cdcl3 fluorescence at mitosis reflects the destruction of the protein. Thus, it behaves as a classic cyclin. This is not the case for cdc2, the level of which remains constant through the cell cycle. Cells carrying a disrupted copy of the c d cl3 + gene fail to accumulate either cdcl3 or cdc2 in the nucleus. Cells carrying a disrupted cdc2+ gene fail to accumulate cdc2 but reveal apparently normal levels of cdcl3. cdcl3 therefore appears to be required to localise cdc2 to the nucleus but not vice versa. The destruction of cdcl3 at mitosis may allow cdc2 to redistribute to the cytoplasm. et al. 1983). The role of cyclins as M phase activators is confirmed by the observation that microinjection of their m R N A causes cells to enter mitosis (Swenson et al. 1986; Pines and Hunt, 1987). This suggests a functional connection between cyclins and the M phase-activating activity known as maturation-promoting factor or MPF (Smith and Ecker, 1971). The oocyte systems in which cyclins and M PF were originally identified, although providing excellent cell cycle synchrony for biochemi cal studies, are not amenable to investigation by either genetics or cytology. By contrast, the fission yeast Schizosaccharomyces pombe has been used extensively as a model system in which to study the genetic control of cell cycle progression (Nurse et al. 1976; Fantes, 1989). Techniques for protein localisation by immunofluorescence microscopy are also well established in S. pombe (Hagan and Hyams, 1988).
Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae
The Journal of Cell Biology, 1998
The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15 , a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 Њ C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14 , DBF2 , and TEM1 , which are functionally related to CDC15 . Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.
Control of Cell Growth and Division in Saccharomyces Cerevisiae
Critical Reviews in Biochemistry and Molecular Biology, 1986
Microbiology School Referee: Harlyn 0. Halvorson Mallosd rUDp Galactose \ UDPGlucose +Glucose -1-P RPE 2H 3-P-GI yce r a t e [mziq Phosph oeno Ip yr uva te PCK CoA L Malate Succi nale s c v :;:-[:; CoA _ ' : + C D KGD Succinyl-CoA 2-oxoglutarate FIGURE 3. Pathways of yeast intermediary metabolism and sugar utilization. Substances commonly used as carbon sources for growth are boxed. Many of the reactions are incompletely described. For example, for kinases, ATP is indicated as a reactant, but ADP is not shown as a product. The existence, function, or direction of several of the indicated reactions is speculative. Gene designations are referred to in the text. Pi indicates inorganic phosphate and 2H indicates a dehydrogenase reaction. (Adapted from Fraenkel, D. G., The Molecular Biology of the Yeast Saccharornyces: Metabolism and Gene Expression. Strathem, J. N., Jones, E. W., and Broach, J. R., Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982, I.) Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by 94.23.205.32 on 05/20/14 For personal use only. Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by 94.23.205.32 on 05/20/14 For personal use only.
Identification of a glycoprotein involved in cell cycle progression in yeast
Journal of Biological Chemistry
The molecular events of start, the regulatory step that commits yeast cells to DNA replication, have recently begun to be investigated. One of the gene products required for completion of start has been found to have a significant structural homology with oncogenes endowed with protein kinase activity. Our experiments provide data on the biosynthetic pathway of a previously identified labile protein (p100, molecular weight 100,000, isoelectric point of approximately 4.8-5) involved in cell cycle progression at start, which appears to be specifically made during the release from cell cycle arrest of a temperature-sensitive mutant (cdc25) of Saccharomyces cerevisiae. On two-dimensional gel, p100 migrates very close to another 100-kDa labile protein (p100*) which behaves as a cell cycle modulated protein with reduced synthesis in G1. Pulse and chase labeling of protein with [35S]methionine suggests that both p100 and p100* are processed to a protein (p115) of slightly higher molecular ...
Molecular and Cellular Biology, 1989
Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans. To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs. This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals.
Molecular and Cellular Biology, 1996
Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with se...